1. Field of the Invention
This invention describes the use of recombinant DNA technology for the design and synthesis of novel, modified interferons. More specifically the invention relates to interferons not known in nature, which are intended for use in viral and neoplastic diseases, and immunosuppressed and immunodeficient conditions.
2. Description of the Prior Art
The interferons are a class of proteins that occur in vertegrates and act as biological regulators of cell function which include increasing resistance to pathogens, limiting cell growth and modulating the immune system. The most studied property of the interferons is their ability to convert cells into an "antiviral state" during which they are more resistant to virus replication (Lengyel, Annual Review of Biochemistry, 51, 251, 1982). In addition to conferring antiviral resistance to target cells, interferon (IFNs) have antiproliferative (antigrowth) properties (Stewart, 1979, The Interferon System, Springer, Berlin). It has clearly been shown that interferons produced naturally act as antiviral and antiproliferative agents (Gresser et al, Biochim. Biophys. Acta, 516, 231, 1978; J. Exp. Med., 144, 1316, 1976).
The IFNs, by virtue of their antigenic, biological and physico-chemical properties, may be divided into three classes: type I, IFN-.alpha. ("leucocyte") and IFN-.beta. ("fibroblast"); and type II, IFN-.gamma. ("immune") (Stewart et al, Nature, 286, 110, 1980). Both genomic DNA and cDNA clones of type I and type II IFNs have been isolated and sequenced, and the potential protein sequences deduced (e.g. Pestka, Arch. Biochem. Biophys, 221, 1, 1983). Whilst in man only one IFN-.beta. and IFN-.gamma. gene are known, human IFN-.alpha. is specified by a multigene family comprising at least 20 genes. The classification of IFN-.beta. and IFN-.alpha. as type I interferons is in part determined by their significant degree of homology, &gt;23% at the protein level (Taniguchi et al, Nature, 285, 547, 1980).
Whilst the mechanism of action of interferons is not completely understood, certain physiological or enzymatic activities respond to the presence of the interferons. These activities include RNA and protein synthesis. Among the enzymes induced by interferons is (2'-5') (A)n synthetase which generates 2'-5' linked oligonucleotides, and these in turn activate a latent endoribonuclease, RNAse L, which cleaves single-stranded RNA, such as messenger RNA (mRNA) and ribosomal RNA (rRNA). Also induced by IFNs is a protein kinase that phosphorylates at least one peptide chain initiation factor and this inhibits protein synthesis (Lengyel, ibid, p. 253). IFNs have been shown to be negative growth regulators for cells by regulation of the (2'-5') An synthetase activity (Creasey et al, Mol. and Cell Biol., 3, 780, 1983). IFN-.beta. was indirectly shown to be involved in the normal regulation of the cell cycle in the absence of inducers through the use of anti-IFN-.beta. antibodies. Similarly, IFNs have been shown to have a role in differentiation (Dolei et al, J. Gen. Virol., 46, 227, 1980) and in immunomodulation (Gresser, Cell. Immunol., 34, 406, 1977). Finally, IFNs may alter the methylation pattern of mRNAs and alter the proportion of fatty acids in membrane phospholipids, thereby changing the rigidity of cell membranes.
These and other mechanisms may respond to interferon-like molecules in varying degrees depending on the structure of the interferon-like polypeptide. Preliminary evidence (UK Patent No. GB 2 090 258A) suggests that members of the multigene IFN-.alpha. family vary in the extent and specificity of their antiviral activity (Pestka, ibid.). For example, combination of IFN-.alpha.A with IFN-.alpha.D resulted in "hybrid" genes which show antiviral properties that are distinct from either parent molecule (Weck et al, Nucl. Acids Res., 9, 6153, 1981; De La Maza et al, J. IFN Res., 3, 359, 1983; Fish et al, Biochem. Biophys. Res. Commun., 112, 537, 1983; Weck et al, Infect. Immuno., 35, 660, 1982). However, hybrid human IFNs with significantly increased human cell activity/specificty have not yet been developed. One Patent has been published describing IFN-.beta./.alpha. hybrids (PCT/US83/00077). This patent describes three examples, none of which have significantly improved activity. The three examples were constructed using two naturally occurring restriction sites. The resulting hybrid interferons were (1) alpha 1 (1-73)-beta (74-166); (2) beta (1-73)-alpha 1 (74-166); and (3) alpha 61A (1-41)-beta (41-166). These three examples differ structurally from the examples of the present invention. These three examples were based upon the accidental location of two restriction sites and not upon the intentionally designed DNA and amino acid sequences of the present invention.
It is envisaged that a modified interferon will display a new advantageous phenotype. The design and synthesis of new interferon-like polypeptides composed of portions of IFN-.beta. and other amino acid sequences is advantageous for the following reasons:
1. New IFNs can be created which show a greater antiproliferative to antiviral activity (and vice versa) resulting from the selective activation of only some of the normal interferon-induced biochemical pathways.
2. The affinity of hybrid or modified IFNs for cell surface receptors will differ from that of naturally occurring interferons. This should allow selective or differential targeting of interferons to a particular cell type, or increased affinity for the receptor--leading to increased potency against a particular virus disease or malignancy.
3. It will be possible to design novel IFNs which have an increased therapeutic index, thus excluding some of the undesirable side effects of natural IFNs which limit their use (Powledge, T. M., Biotechnology, 2, 214, March 1984).
4. Novel IFNs include in the design structures which allow increased stability to proteolytic breakdown during microbial synthesis.
5. Novel IFNs can be designed to increase their solubility or stability in vivo, and prevent non-specific hydrophobic interactions with cells and tissues.
6. Novel IFNs can be designed which are more readily recovered from the microbial supernatant or extract, and more easily purified.